1. Field of the Invention
The present invention relates to the field of surface modification of a substrate so that a biomolecule may be attached to it.
2. Related Art
3. Background
The attachment of biomolecules on a suitable surface has found a variety of bioanalytical applications such as in microarrays, biochemical sensors, etc. In a biological microarray system, “probe” biomolecules such as polynucleotides, oligonucleotides, proteins or peptides are immobilized on a suitable surface. These probes are used to capture other biomolecules (“targets”) that can recognize the probes. For example, in a DNA microarray, immobilized probe polynucleotides or oligonucleotides hybridize to complementary target DNA or DNA fragments in solution, providing sequence information for the targets that can be used to monitor gene expression, map genes, identify bacteria etc. In an immunoarray, immobilized protein antibody or antigen is used to bind to a specific antigen or antibody, respectively, thus making it possible to detect proteins of clinical interest through detection of their binding to the cognate probe.
It is clear that for applications such as those mentioned above, one crucial requirement is appropriate immobilization of probes on a suitable substrate. During the last two decades, a large number of approaches have been developed for immobilization of different biomolecular probes. These strategies may be categorized as pre-synthesized or on-site synthesized, depending on whether the probes are synthesized or extracted and then immobilized on the substrate, or synthesized directly on the substrate.
Pre-synthesized probes are generally either natural products extracted from biological entities or chemically synthesized in a lab; on-site synthesis mainly refers to the solid-phase synthesis of oligonucleotides on an array substrate, such as disclosed in U.S. Pat. No. 5,445,934, issued to Fodor et al. on Aug. 29, 1995, entitled “Array of oligonucleotides on a solid substrate.” In this patent, a photosynthesis strategy is applied to synthesize oligonucleotide probes with different sequences on a glass slide. This patent discloses that linker molecules can be attached to the substrate via carbon-carbon bonds using, for example, (poly)trifluorochloroethylene surfaces, or preferably, by siloxane bonds (using, for example, glass or silicon oxide surfaces). Siloxane bonds with the surface of the substrate may be formed in one embodiment via reactions of linker molecules bearing trichlorosilyl groups.
Methods for immobilizing pre-synthesized probes onto a solid substrate fall into two categories: covalent attachment and simple adsorption. Adsorption of probes onto substrates often takes advantage of electrostatic attraction between substrates and probes. For example, a glass is generally negatively charged when in a neutral or basic solution. It can then adsorb positively charged amine polymers. The polyamine can in turn serve as a support to adsorb other negatively charged probes such as oligos or many proteins. In general, however, simple adsorption does not provide strong enough attachment and thus covalent interaction between substrate and probes is required. One exception of noncovalent adsorption is the strong interaction between biotin and streptavidin, which has been applied for attaching different probes. For example, biotin-labeled probes can be immobilized to streptavidin-modified substrates and form a strong attachment. However, this scheme also requires efficient, strong binding between streptavidin and the substrate.
In reality, covalent attachment of probes onto substrates is more reliable and adopted more often. For covalent attachment, the substrates are modified with suitable reactive groups or linkers for probes; at the same time, the probes carry groups reactive toward the modified substrates and so can form covalent bonds with the reactive groups on the substrates. Modification of substrates with different reactive groups has been well documented. These groups include hydroxyl, carboxyl, amine, aldehyde, hydrazine, epoxide, etc. The modified substrates can then be used to constitute arrays such as DNA or protein microarrays for capturing DNA or protein of interest.
One concern in the modification of substrates is the efficiency of capturing target molecules by probes immobilized on the substrates. When the probes are bound on a flat substrate, the steric crowding between the probes is thought to be responsible for low capture efficiency as observed in some arrays. One strategy to overcome this shortcoming is to bind the probes in a 3D substrate or a substrate modified with a 3D support. Application of 3D polymer matrices for immobilizing probes has been disclosed in U.S. Pat. No. 6,391,937, issued to Beuhler et al. on May 21, 2002, entitled “Polyacrylamide hydrogels and hydrogel arrays made from polyacrylamide reactive prepolymers.” As disclosed there, a hydrogel of either polyacrylate or urea containing reactive groups for probes is deposited onto glass substrates.
U.S. Pat. No. 7,026,014, issued to Luzinov et al. on Apr. 11, 2006, entitled “Surface modification of substrates,” discloses a process that includes applying polymer comprising multiple epoxy groups and having a molecular weight of at least about 2000 to the surface of a substrate. Other epoxy groups in the anchoring layer, not utilized in forming the layer, may be used to graft surface modifying materials to the surface. For instance, macromolecules, biomolecules, polymers, and polymerization initiators may be grafted to the surface via the anchoring layer.
Pompe, et al., “Maleic Anhydride Copolymers—A Versatile Platform for Molecular Biosurface Engineering,” Biomacromolecules 4:1072-1079 (2003) discloses the use of different maleic anhydride copolymers such as PEMA, poly(ethylene-alt-maleic anhydride), which were covalently bound to SiO2 surfaces. The copolymer solutions were applied to substrate surfaces which had been freshly oxidized and thereafter surface modified with 3-aminopropyl-dimethylethoxy-silane. Stable covalent binding of the polymer films was achieved by annealing at 120° C. for generation of imide bonds with the amino-silane on the SiO2 substrate. Amino functionalization of the substrates was achieved on SiO2 surfaces by silanization. Polymer substrates were amino-functionalized by low-pressure plasma treatment in ammonia atmospheres. Biomolecules (e.g., fibronectin) were attached to the maleic anhydride copolymers through reactivity of the anhydride function toward amines. This process uses an amino-silane, and does not suggest the use of a polyamine. As a silanization process, such a method involves organic solvents and may have variable results due to reproducibility problems in the process.
The present methods and materials have particular application to microarrays of nucleic acid or peptide probes immobilized on a substrate, and particularly to microarrays which employ structures added after the polymer surface treatment, such as would be added by etching and/or photolithography in a microfabrication process.
DNA microarrays have become essential devices in molecular biology as they allow high-throughput analysis of transcriptional profiling (REF 1) and medical diagnostics (REF 2). The success of DNA microarrays has also inspired a recent trend of combining microelectromechanical systems (MEMS) with biomolecules (BioMEMS). By integrating microarrays and detection systems into a miniaturized single device, BioMEMS has the potential to revolutionize the future practice of medicine and health care (REF 3). Surface chemistries developed for conventional microarrays, however, are not easily adopted in these BioMEMS systems. The incompatibility of biomolecules and microfabrication is caused mainly by two factors. First, the microfabrication process often involves elevated temperatures and harsh chemical and physical treatments that can denature or damage biomolecules; second, DNA immobilization chemistries always start with a strong acid cleaning such as Piranha or a strong base cleaning such as RCA to activate the substrates, a harsh condition that the components (e.g., sensors) of a MEMS device can rarely survive. It is thus very challenging to integrate biomolecules into common microfabrication processes (REF 4).